BACKGRUND: Despite new therapeutic approaches, asbestos-related diseases such as pleural mesothelioma (PM) and lung cancer (LC) remain incurable and resistant to conventional therapies. The lack of accurate models that reflect the biological characteristics of primary tumors makes PM difficult to study. Patient-derived organoids (PDOs) closely replicate the genotypic and phenotypic characteristics of their parental tumors, making them ideal preclinical models. In this context, malignant pleural effusions (MPE) provide a non-invasive and valuable source for generating PDOs, offering a platform to evaluate therapeutic responses and resistance-linked signatures. METHODS: Isolated cells from MPEs and pleural biopsies (PBs) were used to establish PDOs for PM and LC. After standardizing culture conditions, the PDOs were characterized morphologically and histologically. LC-PDOs were tested for drug response, while PM-PDOs underwent drug testing, transcriptomic analysis compared to benign reactive mesothelial (RM) counterpart, and miRNome analysis, including bioinformatic assessment of miRNA-target gene regulatory networks in response to treatments. RESULTS: The PDOs preserved the morphological and histopathological characteristics of the original tumors. Regarding culture conditions, PM-PDOs grew preferentially in Matrigel, while LC-PDOs grew either in Matrigel or Hydrogel. Furthermore, PM-PDOs demonstrated optimal growth with EGF alone, whereas stemness supplements were essential for the cultivation of LC-PDOs. In addition, PM-PDOs establishment from MPE yielded higher success rates from epithelioid histotype cases than biphasic cases. As expected, PDOs exhibited differential responses to anticancer treatments, highlighting the potential of personalized medicine. In particular, tumor-associated macrophages (TAMs) in PM-PDOs influenced the treatment response. Transcriptomic analysis shows differential genes expression between PM- and RM-PDOs. Indeed, pathways enrichment underscored mitochondrial dysfunction and TNF-α downregulation as a hallmark of malignancy in PM-PDOs. MiRNA profiling identified key miRNAs and target hub genes emerged as critical players in treatment response and resistance, highlighting their potential as predictive biomarkers and therapeutic targets. CONCLUSIONS: This study demonstrates the successful establishment of PDOs from MPE cells in LC and PM, positioning MPE as a non-invasive and valuable source for generating biologically relevant tumor models. These models offer a platform for evaluating therapeutic responses and elucidating the roles of specific miRNAs in treatment resistance.
INTRODUZIONE: Nonostante i nuovi approcci terapeutici, le malattie correlate all'amianto, come il mesotelioma pleurico (MP) e il carcinoma polmonare (CP), rimangono incurabili e resistenti alle terapie convenzionali. La mancanza di modelli accurati che riflettano le caratteristiche biologiche dei tumori primari rende difficile lo studio del MP. Gli organoidi derivati da pazienti (PDOs) replicano fedelmente le caratteristiche genotipiche e fenotipiche dei tumori parentali, rendendoli modelli preclinici ideali. In questo contesto, i versamenti pleurici maligni (VPM) rappresentano una fonte non invasiva e preziosa per generare PDOs, offrendo una piattaforma per valutare le risposte terapeutiche e i meccanismi di resistenza. METODI: Le cellule isolate da VPM e biopsie pleuriche (PB) sono state utilizzate per stabilire PDOs per MP e CP. Dopo la standardizzazione delle condizioni di coltura, i PDOs sono stati caratterizzati morfologicamente e istologicamente. I PDOs derivati da CP (CP-PDOs) sono stati testati per la risposta ai farmaci, mentre i PDOs derivati da MP (MP-PDOs) sono stati sottoposti a test farmacologici, analisi trascrittomica rispetto alla controparte mesoteliale reattiva benigna (RM) e analisi del miRNoma, inclusa la valutazione bioinformatica delle reti regolatorie miRNA-gene target in risposta ai trattamenti. RISULTATI: I PDOs hanno mantenuto le caratteristiche morfologiche e istopatologiche dei tumori originali. Per quanto riguarda le condizioni di coltura, i MP-PDOs crescevano preferenzialmente in Matrigel, mentre i CP-PDOs crescevano sia in Matrigel che in Hydrogel. Inoltre, i MP-PDOs hanno mostrato una crescita ottimale utilizzando il solo fattore EGF, mentre i fattori di staminalità sono stati essenziali per la formazione di CP-PDOs. L'ottenimento di MP-PDOs da VPM ha avuto maggior successo nei casi di istotipo epitelioide rispetto ai casi di istotipo bifasico. Come previsto, i PDOs hanno risposto differentemente ai trattamenti antitumorali, evidenziandone il potenziale uso nell'ambito della medicina personalizzata. In particolare, i macrofagi associati al tumore (TAM) nei MP-PDOs hanno influenzato la risposta ai trattamenti. L'analisi trascrittomica ha evidenziato differenze nell'espressione genica tra MP-PDOs e RM-PDOs. Il pathays enrichment, ha riportato la disfunzione mitocondriale e la downregolazione del TNF-α come segni distintivi del MP. L'analisis del miRNoma ha identificato i miRNA e geni hub coinvolti nella risposta e resistenza ai trattamenti, evidenziandone il potenziale come biomarcatori predittivi e bersagli terapeutici. CONCLUSIONI: Questo studio dimostra il successo nell'ottenimento di PDOs da cellule di VPM nei casi di CP e MP, confermando il VPM come una fonte non invasiva e preziosa, per generare modelli tumorali biologicamente rilevanti. Questi modelli offrono una piattaforma per valutare le risposte terapeutiche e chiarire il ruolo specifico dei miRNA nella resistenza ai trattamenti.
Patient-derived organoids from pleural effusion: A 3D platform for malignant pleural mesothelioma
VOLPINI, LUCA
2025
Abstract
BACKGRUND: Despite new therapeutic approaches, asbestos-related diseases such as pleural mesothelioma (PM) and lung cancer (LC) remain incurable and resistant to conventional therapies. The lack of accurate models that reflect the biological characteristics of primary tumors makes PM difficult to study. Patient-derived organoids (PDOs) closely replicate the genotypic and phenotypic characteristics of their parental tumors, making them ideal preclinical models. In this context, malignant pleural effusions (MPE) provide a non-invasive and valuable source for generating PDOs, offering a platform to evaluate therapeutic responses and resistance-linked signatures. METHODS: Isolated cells from MPEs and pleural biopsies (PBs) were used to establish PDOs for PM and LC. After standardizing culture conditions, the PDOs were characterized morphologically and histologically. LC-PDOs were tested for drug response, while PM-PDOs underwent drug testing, transcriptomic analysis compared to benign reactive mesothelial (RM) counterpart, and miRNome analysis, including bioinformatic assessment of miRNA-target gene regulatory networks in response to treatments. RESULTS: The PDOs preserved the morphological and histopathological characteristics of the original tumors. Regarding culture conditions, PM-PDOs grew preferentially in Matrigel, while LC-PDOs grew either in Matrigel or Hydrogel. Furthermore, PM-PDOs demonstrated optimal growth with EGF alone, whereas stemness supplements were essential for the cultivation of LC-PDOs. In addition, PM-PDOs establishment from MPE yielded higher success rates from epithelioid histotype cases than biphasic cases. As expected, PDOs exhibited differential responses to anticancer treatments, highlighting the potential of personalized medicine. In particular, tumor-associated macrophages (TAMs) in PM-PDOs influenced the treatment response. Transcriptomic analysis shows differential genes expression between PM- and RM-PDOs. Indeed, pathways enrichment underscored mitochondrial dysfunction and TNF-α downregulation as a hallmark of malignancy in PM-PDOs. MiRNA profiling identified key miRNAs and target hub genes emerged as critical players in treatment response and resistance, highlighting their potential as predictive biomarkers and therapeutic targets. CONCLUSIONS: This study demonstrates the successful establishment of PDOs from MPE cells in LC and PM, positioning MPE as a non-invasive and valuable source for generating biologically relevant tumor models. These models offer a platform for evaluating therapeutic responses and elucidating the roles of specific miRNAs in treatment resistance.File | Dimensione | Formato | |
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https://hdl.handle.net/20.500.14242/202958
URN:NBN:IT:UNIVPM-202958